With increasing number of completely sequenced genomes, more and more information needs to be analyzed, such as enormous encoding messages of these genome sequences, controlling elements of different genes dispersed in genomes, and their biological function involved. Sequence analysis methods developed from functional genome research mainly includes microarrays and quantitative assay based on isolated DNA sequence.
Most used microarray platforms are oligonucleotide arrays. In this technology, different oligos representing different segments of the genome are immobilized on a vector's surface, and then the array is hybridized with samples proportionally. Thus the strength of hybridization signal defines target sequence relative abundance. The major advantage of oligonucleotide arrays is the convenience to handle, and the ability of its massively parallel operation. However, global analysis of gene expression profile in tissue or cells is difficult to be obtained with microarray technology. In addition, microarray technology needs to predetermine gene probes to be synthesized. Thus some unknown and less abundant expressed genes could be missed in the assay. Furthermore, cross hybridization phenomena may influence the accuracy of the results.
Currently the most commonly used method is quantitative assay based on isolated DNA sequence. In this type of method, originally used method is SAGE (Serial analysis of gene expression). This method is to analyze expression status of groups of genes in certain tissue or cell types based on sequencing technology. The prevailing method is a polony sequencing method used in Church's group. This technology is a revised version of SAGE. Its sequencing process is illustrated in FIG. 1, including the following steps: Step 11, processing DNA template into DNA tag by random sonication or molecular biology method; Step 12, DNA tag is immobilized on microbeads by amplification through emulsion PCR (Polymerase Chain Reaction), and these microbeads are then embedded in agarose and tightly arrayed on glass surface. Step 13, parallel sequencing of DNA tags on microbeads via Ligation. First, hybridize between single strand DNA and sequence anchor, and then ligase select base through the ligation of 4 fluorescent labeled oligonucleotide. This process will produce sequencing signal. In this step, since sequencing anchor used is immobilized anchor, bases that can be called is less than 7 by ligation reaction. Step 14, sequencing signal are collected, images are processed and bases are called. Correctly ligated base can be imaged by fluorescence label.
In the above said technology, bases to be sequenced by sequencing anchor are generally within 7 bases from sequencing anchor. Only short DNA tags can be sequenced. Since sequences obtained are relatively too short, it is hard to map obtained sequence back into genome sequences. Thus sequencing information is difficult to be used efficiently. Furthermore, the immobilization of micro-beads is through embedding beads in gel on surface of slides. In this way, density of micro-beads can't be very high. Throughput and reaction efficiency are difficult to improve. In addition, when 4 color fluorescence labeling system is adapted, signal strength difference among different fluorescence could affect the result. Different signal can even be masked by background. Therefore sequencing results could be inaccurate or misleading.
Thus a new sequencing method is needed to enlarge the application area of DNA sequencing. This new method should be higher throughput, reaction efficiency and accuracy.